Compositions comprising sulforaphane or a sulforaphane precursor and magnesium

ABSTRACT

The invention relates to the combination of a sulforaphane precursor, an enzyme capable of converting the sulforaphane precursor to sulforaphane, an enzyme potentiator, and magnesium or a salt or complex thereof. The invention also relates to the combination of a sulforaphane or a derivative thereof and magnesium or a salt or complex thereof. The invention also relates to the combination of a broccoli extract or powder and magnesium or a salt or complex thereof. The invention provides compositions and methods relating to these combinations.

This application claims priority to the following applications, each ofwhich is incorporated by reference in its entirety: U.S. ProvisionalPatent Application No. 61/668,328, filed on Jul. 5, 2012; U.S.Provisional Patent Application No. 61/668,342, filed on Jul. 5, 2012;U.S. Provisional Patent Application No. 61/668,386, filed on Jul. 5,2012; U.S. Provisional Patent Application No. 61/668,396, filed on Jul.5, 2012; U.S. Provisional Patent Application No. 61/668,364, filed onJul. 5, 2012; U.S. Provisional Patent Application No. 61,668,374, filedon Jul. 5, 2012; and U.S. Provisional Patent Application No. 61/794,417,filed on Mar. 15, 2013.

FIELD OF THE INVENTION

The present invention relates to the combination of a sulforaphaneprecursor, an enzyme capable of converting the sulforaphane precursor tosulforaphane, an enzyme potentiator, and magnesium or a salt or complexthereof. The present invention also relates to the combination of asulforaphane or a derivative thereof and magnesium or a salt or complexthereof. The present invention also relates to the combination of abroccoli extract or powder and magnesium or a salt or complex thereof.The present invention provides compositions and methods relating tothese combinations.

BACKGROUND OF THE INVENTION

The use of natural products is becoming increasingly popular with humansand companion animals. Some of these natural products are beingincorporated into dietary supplements and medical foods. There is a needin the art for supplements which are useful as chemoprotective and/orantioxidant agents. In addition, there is a need in the art forpharmaceutical compositions and dietary supplements which are useful forconditions and disorders associated with cardiovascular health.

More than 1 in 3 (83 million) U.S. adults currently live with one ormore types of cardiovascular disease. Respectively, heart disease andstroke are the first and third leading causes of death in the US with anestimated 935,000 heart attacks and 795,000 strokes occurring each year.Nearly 68 million adults have high blood pressure, and about half haveuncontrolled hypertension. Additionally, an estimated 71 million adultshave high cholesterol (i.e., high levels of low-density lipoproteincholesterol), with an estimated ⅔ of this population having uncontrolledhigh cholesterol. These health problems are among the most widespreadand costly of chronic illnesses in the United States today, accountingfor approximately one out of every six dollars spent on healthcare. Atthe “heart” of cardiovascular disease is endothelial dysfunction. Theendothelium, a thin membrane of flattened cells that line all bloodvessels, is one of the largest organs in the body. The vascularendothelia play vital roles in blood flow, blood pressure regulation,coagulant and anticoagulant activity as well as control the selectiveadhesion and tissue migration of white blood cells. Endothelialdysfunction is the greatest underlying cause of atherosclerosis,hypertension, and inflammatory vascular diseases that lead to many ofthe catastrophic heart, circulatory, renal, and neurological conditionsaffecting the population. Endothelial dysfunction which invariably leadsto major cardiovascular events, is most often due to a chronicinflammatory state brought on by conditions such as diabetes (both type2 and insulin dependent diabetes), immune disorders (eg. lupus andrheumatoid arthritis), as well as a host of other chronic diseases.These conditions are positively associated with increased levels ofspecific inflammatory markers which include C-Reactive Protein (CRP),Interleukin-8 (IL-8), and monocyte chemoattractant protein (MCP-1).Recommendations to control endothelial dysfunction include lifestylemodifications such as eating a healthy diet, exercise, weight loss,avoidance of tobacco and second hand smoke, lowering cholesterol andcontrolling diabetes.

Chemoprotection through the use of natural products is evolving as asafe, effective, inexpensive, easily accessible, and practical means toprevent or reduce the occurrence of many conditions affecting humans anddomesticated animals. It is known that carcinogens which can damagecells at the molecular level are often ingested and inhaled as non-toxicprecursors. These non-toxic precursors may then convert intocarcinogenic substances in the body. Chemoprotective agents, such asnatural substances which can activate detoxifying enzymes or theirco-factors, can counteract and allow for the elimination of carcinogens.These same natural substances can potentiate other naturally existingdefenses such as the immune system.

Atherosclerosis results from a number of different inflammatorycascades. Evidence points to monocyte-derived macrophages as being oneof the key cell types involved in endothelial inflammation.Interleukin-8 (IL-8) is responsible for recruiting monocytes andmacrophages and monocyte chemoattractant protein-1 (MCP-1) plays a rolein migration and infiltration of these cells. These proteins have beenfound to be significantly increased in the arterial atherosclerotic walland worse, recruit additional inflammatory cells forming a viciouscycle. Mechanisms that can suppress both biomarkers (MCP-1 and IL-8)would diminish inflammation and subsequent atherosclerosis. The role ofIL-8 in cardiovascular disease is discussed in Apostolakis et al.Cardiovasc Res, 2009, 84(3): 353-360; and Aukrust et al. ArteriosclerThromb Vasc Biol, 2008, 28:1909-1919. The role of MCP-1 incardiovascular disease is discussed in Niu et al., Clin Sci (Loud),2009, 117(3):95-109, and Hoogeveen et al, Atherosclerosis, 2005, 183(2):301-307. MCP-1 and IL-8 is associated with a number of otherinflammatory and vascular diseases and conditions. Examples of diseasesand conditions associated with MCP-1 and IL-8 include, but are notlimited to atherosclerosis, inflammatory bowel disease, inflammatorylung disease, chronic liver disease, inflammatory rheumatic disease,gingivitis, asthma, psoriasis, Alzheimer's disease, ischemic heartdisease, acute coronary syndrome, arterial injury, and arteriogenesis.

Some natural products have antioxidant activity. Oxidative stress playsa major role in aging, the progression of neurodegenerative diseases aswell as physiological trauma, such as ischemia. Antioxidant agents canreduce or inhibit the oxidation of vital biomolecules and may play arole in treating, preventing, or reducing the occurrence of conditionsaffected by oxidative stress.

An example of a natural product thought to have chemoprotective andantioxidant properties is sulforaphane. Sulforaphane is an organosulfurcompound which is also known as 1-isothiocyanato-4-methylsulfinylbutane.The sulforaphane precursor, glucoraphanin, can be obtained fromvegetables of the Brassicaceae family, such as broccoli, brusselsprouts, and cabbage. However, copious amounts of vegetables must beconsumed in order to obtain levels adequate for chemoprevention.Glucoraphanin is converted into sulforaphane by a thioglucosidase enzymecalled myrosinase, which occurs in a variety of exogenous sources suchas Brassicaceae vegetables and endogenously in the gut microflora.However, upon ingestion of glucoraphanin, not all animals are capable ofachieving its conversion to sulforaphane, most likely due to variationsin microflora populations and overall health. In addition, in acidicenvironments such as the stomach, glucoraphanin can be converted toinert metabolites. The active metabolite, sulforaphane induces nuclearerythroid-2-related factor (Nrf2) which, in turn, upregulates theproduction of Phase II detoxification enzymes and cytoprotective enzymessuch as glutathione S-transferases, NAD(P)H:quinine oxidoreductase(NQO1), and heme-oxygenase-1 (HO-1). Sulforaphane has been thought toinduce the production of these enzymes without significantly changingthe synthesis of P-450 cytochrome enzymes. The upregulation of Phase IIenzymes is thought to play a role in a variety of biological activities,including the protection of the brain from cytotoxicity, the protectionof the liver from the toxic effects of fat accumulation, and thedetoxification of a variety of other tissues.

Sulforaphane and its precursor glucoraphanin have been studiedextensively. Shapiro et al. (Nutrition and Cancer, (2006), Vol. 55(1),pp. 53-62) discuss a clinical Phase I study determining the safety,tolerability, and metabolism of broccoli sprout glucosinolates andisothiocyanates. Shapiro et al. discuss a placebo-controlled,double-blind, randomized clinical study of sprout extracts containingeither glucosinolates such as glucoraphanin or isothiocyanates such assulforaphane in healthy human subjects. The study found thatadministration of these substances did not result in systematic,clinically significant, adverse effects. Ye et al., (Clinica ChimicaActa, 200, 316:43-53) discuss the pharmacokinetics of broccoli sproutisiothiocyanates in humans.

Magnesium is a mineral that is important for many systems in the body,including muscles and nerves. Magnesium is vital for the metabolism ofadenosine triphosphate (ATP) and is necessary for protein, fat, andnucleic acid synthesis. Adequate magnesium levels are important for theprevention of hypertension, which is a major cause of endothelialdysfunction. Magnesium is also important for calcium absorption, calciumregulation, and bone mineralization. While there are many magnesium saltforms that can augment dietary magnesium intake, the chelated form ofmagnesium (magnesium chelate) has been found to provide improvedbioavailability of magnesium while providing a decreased side effectprofile. For example, compared to salts of magnesium such as magnesiumsulfate, magnesium chelates have been found to exert a decreasedlaxative effect.

Other supplements are also known to be beneficial, especially in thearea of cardiovascular health. Vitamin K2, also known as menaquinone, isa fat-soluble vitamin that has been shown to be essential for calciumregulation in the body. Menaquinone occurs naturally in variousmolecular forms, characterized by the number of isoprene side chains(n), and designated as menaquinone-n (MK-n). MK-7 is most readilyobtained through the fermentation of soybeans to make natto. Low levelsof vitamin K2 are associated with vascular calcification, includingcalcification of atherosclerotic plaques, and osteopenic andosteoporotic conditions. Supplementation with vitamin K2 may decreasecholesterol plaque calcification, decrease arterial stiffness, andimprove bone mineralization. Zhang et al. (Proc. Natl. Acad. Sci.,(1994), Vol. 91, pp. 3147-3150) discuss a study in Sprague-Dawley ratsto determine the anticarcinogenic activities of sulforaphane andstructurally related synthetic norbornyl isiothiocyanates. The studydetermined that administration of sulforaphane was effective in blockingthe formation of mammary tumors.

Cornblatt et al. (Carcinogenesis, (2007), Vol. 38(7): pp. 1485-1490)discuss a study in Sprague-Dawley rats to determine the effect ofsulforaphane in chemoprevention in the breast. The study determined thatoral administration of sulforaphane resulted in a 3-fold increase inNAD(P)H:quinine oxidoreductase (NQO1) enzymatic activity and a 4-foldelevated immunostaining of the heme oxygenase-1 (HO-1) enzyme in themammary epithelium.

European Patent Application No. 2 213 280 discloses formulationscomprising glucosinolates such as glucoraphanin and myrosinase, whereinthe formulation is encapsulated or coated.

All references cited herein are incorporated by reference in theirentirety.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising: (i) asulforaphane precursor, preferably glucoraphanin; (ii) an enzyme capableof converting the sulforaphane precursor to sulforaphane, preferably aglucosidase enzyme, more preferably a thioglucosidase enzyme, and mostpreferably myrosinase; (iii) an enzyme potentiator, preferably ascorbicacid; and (iv) magnesium or a salt or complex thereof. The presentinvention also provides a method of treating, preventing, reducing theoccurrence of, decreasing the symptoms associated with, and/or reducingsecondary recurrences of, conditions associated with the endothelium andcardiovascular system, comprising administering to the subject: (i) asulforaphane precursor, (ii) an enzyme capable of converting thesulforaphane precursor to sulforaphane, (iii) an enzyme potentiator, and(iv) magnesium or a salt or complex thereof. The present invention alsoprovides a method of decreasing levels or decreasing gene expression ofinterleukin-8 (IL-8) and/or monocyte chemoattractant protein-1 (MCP-1)in a subject, comprising administering to the subject: (i) asulforaphane precursor, (ii) an enzyme capable of converting thesulforaphane precursor to sulforaphane, (iii) an enzyme potentiator, and(iv) magnesium or a salt or complex thereof. The present invention alsoprovides a method of treating, preventing, reducing the occurrence of,decreasing the symptoms associated with, and/or reducing secondaryrecurrences of a disease or condition associated with elevated levels ofinterleukin-8 (IL-8) and/or monocyte chemoattractant protein-1 (MCP-1),comprising administering to the subject: (i) a sulforaphane precursor,(ii) an enzyme capable of converting the sulforaphane precursor tosulforaphane, (iii) an enzyme potentiator, and (iv) magnesium or a saltor complex thereof.

The present invention provides a composition comprising: (i)sulforaphane or a derivative thereof, and (ii) magnesium or a salt orcomplex thereof. The present invention also provides a method oftreating, preventing, reducing the occurrence of, decreasing thesymptoms associated with, and/or reducing secondary recurrences of,conditions associated with the endothelium and cardiovascular system ina subject, comprising administering to the subject: (i) sulforaphane ora derivative thereof, and (ii) magnesium or a salt or complex thereof.The present invention also provides a method of decreasing levels ordecreasing gene expression of interleukin-8 (IL-8) and/or monocytechemoattractant protein-1 (MCP-1) in a subject, comprising administeringto the subject, comprising administering to the subject: (i)sulforaphane or a derivative thereof, and (ii) magnesium or a salt orcomplex thereof. The present invention also provides a method oftreating, preventing, reducing the occurrence of, decreasing thesymptoms associated with, and/or reducing secondary recurrences of adisease or condition associated with elevated levels of interleukin-8(IL-8) and/or monocyte chemoattractant protein-1 (MCP-1), comprisingadministering to the subject: (i) sulforaphane or a derivative thereof,and (ii) magnesium or a salt or complex thereof.

The present invention provides a composition comprising: (i) a broccoliextract or powder, and (ii) magnesium or a salt or complex thereof. Thepresent invention also provides a method of treating, preventing,reducing the occurrence of, decreasing the symptoms associated with,and/or reducing secondary recurrences of, conditions associated with theendothelium and cardiovascular system in a subject, comprisingadministering to the subject: (i) a broccoli extract or powder, and (ii)magnesium or a salt or complex thereof. The present invention alsoprovides a method of decreasing levels or decreasing gene expression ofinterleukin-8 (IL-8) and/or monocyte chemoattractant protein-1 (MCP-1)in a subject, comprising administering to the subject, comprisingadministering to the subject: (i) a broccoli extract or powder, and (ii)magnesium or a salt or complex thereof. The present invention alsoprovides a method of treating, preventing, reducing the occurrence of,decreasing the symptoms associated with, and/or reducing secondaryrecurrences of a disease or condition associated with elevated levels ofinterleukin-8 (IL-8) and/or monocyte chemoattractant protein-1 (MCP-1)in a subject, comprising administering to the subject: (i) a broccoliextract or powder, and (ii) magnesium or a salt or complex thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the conversion of glucoraphanin at 38° C.without ascorbic acid, as described in Example 4.

FIG. 2 is a graph showing the conversion within about 10 minutes at 38°C. as a function of ascorbic acid concentration, as described in Example4.

FIG. 3 is a graph showing the conversion to sulforaphane within 30minutes at 38° C. and 1 mM ascorbic acid, as described in Example 4.

FIG. 4 is a graph showing the conversion of glucoraphanin tosulforaphane in simulated intestinal fluid, as described in Example 5.

FIG. 5 is a graph showing the results of the experiment described inExample 6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the combination of a sulforaphaneprecursor, an enzyme capable of converting the sulforaphane precursor tosulforaphane, an enzyme potentiator, and magnesium or a salt or complexthereof. The present invention also relates to the combination ofsulforaphane or a derivative thereof and magnesium or a salt or complexthereof. The present invention also relates to the combination of abroccoli extract or powder and magnesium or a salt or complex thereof.The present invention also relates to the use of magnesium or a salt orcomplex thereof, with a mixture of one or more of the following:sulforaphane precursor, sulforaphane or a derivative thereof, andbroccoli extract. The present invention provides compositions relatingto these combinations.

The present invention also provides methods comprising administeringthese combinations. In some embodiments, the combination may beadministered for treating, preventing, reducing the occurrence of,decreasing the symptoms associated with, and/or reducing secondaryrecurrences of a disease or condition associated with the endotheliumand cardiovascular system in a subject. In some embodiments, thecombination may be administered for decreasing levels or decreasing geneexpression of interleukin-8 (IL-8) and/or monocyte chemoattractantprotein-1 (MCP-1) in a subject. In some embodiments, the combination maybe administered for treating, preventing, reducing the occurrence of,decreasing the symptoms associated with, and/or reducing secondaryrecurrences of a disease or condition associated with elevated levels ofinterleukin-8 (IL-8) and/or monocyte chemoattractant protein-1 (MCP-1)in a subject.

Sulforaphane is also known as 1-isothiocyanato-4-methylsulfinylbutane.Derivatives of sulforaphane include, but are not limited tosulfoxythiocarbamate analogues of sulforaphane, 6-methylsulfinylhexylisothiocyanate (6-HITC), and compounds which comprise the structure ofsulforaphane with different side chains and/or various lengths ofspacers between the isothiocyanato and sulfoxide groups. Examples ofderivatives of sulforaphane include those described in the followingreferences, each of which is incorporated herein by reference: Hu etal., Eur J Med Chem, 2013, 64:529-539; Ahn et al., Proc Natl Acad SciUSA, 2010, 107(21):9590-9595; and Morimistu et al., J. Biol. Chem. 2002,277:3456-3463, and Baird et al., Arch Toxicol, 2011, 85(4):241-272.

In some embodiments, the composition comprises sulforaphane or aderivative thereof, preferably sulforaphane, in an amount of about 1 μgto about 10 g, preferably about 3 μg to about 5 g, preferably about 5 μgto about 1000 mg, preferably about 7 μg to about 750 mg, more preferablyabout 10 μg to about 500 mg, and most preferably about 100 μg to about100 mg. In some embodiments, compositions suitable for human usecomprise about 1 mg to about 20 mg.

In some embodiments, the methods of the present invention compriseadministration of sulforaphane or a derivative thereof to a subject,preferably sulforaphane, in an amount of about 1 μg to about 10 g,preferably about 3 μg to about 5 g, preferably about 5 μg to about 1000mg, preferably about 7 μg to about 750 mg, more preferably about 10 μgto about 500 mg, and most preferably about 100 μg to about 100 mg. Insome embodiments wherein the subject is a human, the method comprisesadministration of about 1 mg to about 20 mg. In some embodiments, themethods of the present invention comprise administration of sulforaphaneor a derivative thereof to a subject, preferably sulforaphane, in anamount of about 0.01 μg/kg to about 0.2 g/kg, preferably about 0.05μg/kg to about 0.07 g/kg, more preferably about 0.07 μg/kg to about 15mg/kg, more preferably about 0.1 μg/kg to about 11 mg/kg, and mostpreferably about 0.2 μg/kg to about 7 mg/kg. In some preferredembodiments wherein the subject is a human, the method comprisesadministration of about 2 μg/kg to about 2 mg/kg, and more preferablyabout 0.01 mg/kg to about 0.3 mg/kg. The above amounts may refer to eachdosage administration or a total daily dosage. The total daily dosagerefers to the total amount of a compound or ingredient which isadministered to a subject in a twenty-four hour period.

In some embodiments, the method comprises administration of more thanone of a sulforaphane or a derivative thereof. In some embodiments, thecompositions comprise more than one of a sulforaphane or a derivativethereof. For example, the methods or composition may comprise bothsulforaphane and one or more derivatives thereof, or two or morederivatives. In some embodiments wherein the method or compositioncomprise more than one of a sulforaphane or a derivative thereof, theabove amounts may refer to the amount of each sulforaphane or aderivative thereof, or the total amount of the more than onesulforaphane or derivative thereof.

The term “sulforaphane precursor” refers to any compound, substance ormaterial which can be used to produce sulforaphane. In preferredembodiments, the sulforaphane precursor comprises a compound which canbe converted or metabolized to sulforaphane, preferably by an enzyme. Insome preferred embodiments, the sulforaphane precursor comprisesglucoraphanin. Glucoraphanin is a glucosinolate which is also known as4-methylsulfinylbutyl glucosinolate and1-S-[(1E)-5-(methylsulfinyl)-N-(sulfonatooxy)pentanimidoyl]-1-thio-β-D-glucopyranose.

In some embodiments, the composition comprises about 1 μg to about 10 g,preferably about 250 μg to about 5 g, more preferably about 500 μg toabout 2000 mg, even more preferably about 1 mg to about 750 mg, evenmore preferably about 1.5 mg to about 250 mg, even more preferably about2 mg to about 100 mg, and most preferably about 3 mg to about 75 mg ofthe sulforaphane precursor, preferably glucoraphanin. In someembodiments, compositions suitable for human use comprise about 3.5 mgto about 50 mg of the sulforaphane precursor, preferably glucoraphanin.

In some embodiments, the method comprises administering the sulforaphaneprecursor, preferably glucoraphanin to a subject, in an amount of about1 μg to about 10 g, preferably about 250 μg to about 5 g, morepreferably about 500 μg to about 2000 mg, even more preferably about 1mg to about 750 mg, even more preferably about 1.5 mg to about 250 mg,even more preferably about 2 mg to about 100 mg, and most preferablyabout 3 mg to about 75 mg. In some embodiments wherein the subject is ahuman, the method comprises administration of about 3.5 mg to about 50mg. In some embodiments, the method comprises administering an amount ofsulforaphane precursor to a subject in an amount of about 1 μg/kg toabout 1000 mg/kg, preferably about 5 μg/kg to about 500 mg/kg, morepreferably about 7.5 μg/kg to about 100 mg/kg, even more preferablyabout 10 μg/kg to about 25 mg/kg, and most preferably about 25 μg/kg toabout 10 mg/kg. In some embodiments wherein the subject is a human, themethod comprises administration of about 50 μg/kg to about 800 μg/kg.The above amounts may refer to each dosage administration or a totaldaily dosage.

In some embodiments, the method comprises administration of more thanone sulforaphane precursor. In some embodiments, the compositioncomprises more than sulforaphane precursor. In some embodiments whereinthe method or composition comprises more than one sulforaphaneprecursor, the above amounts may refer to the amount of eachsulforaphane precursor, or the total amount of the sulforaphaneprecursors.

The sulforaphane precursor may be converted or metabolized tosulforaphane. In some embodiments, the sulforphane precursor isconverted to sulforaphane by an enzyme. In some embodiments, the enzymecapable of converting the sulforaphane precursor to sulforaphanecomprises a glucosidase enzyme, preferably a thioglucosidase enzyme, andmore preferably myrosinase. Myrosinase is also known as thioglucosideglucohydrolase.

In some embodiments, the composition comprises the enzyme in an amountof about 1 pg to about 1 ug, preferably about 50 pg to about 500 ng, andmost preferably about 1 ng to about 150 ng. In some embodiments,compositions suitable for human use comprise about 5 ng to about 75 ngof the enzyme.

In some embodiments, the method comprises administering the enzyme,preferably myrosinase, in an amount of about 1 pg to about 1 μg,preferably about 50 pg to about 500 ng, and most preferably about 1 ngto about 150 ng. In some embodiments wherein the subject is a human, themethod comprises administration of about 5 ng to about 75 ng of theenzyme. In some embodiments, the method comprises administering theenzyme to a subject in an amount of about 0.02 pg/kg to about 0.02ug/kg, preferably about 0.7 pg/kg to about 7 ng/kg, and most preferablyabout 0.02 ng/kg to about 2 ng/kg. In some preferred embodiments whereinthe subject is a human, the method comprises administration of about 0.1ng/kg to about 1 ng/kg. The above amounts may refer to each dosageadministration or a total daily dosage.

In some embodiments, the method comprises administration of more thanone enzyme capable of converting the sulforaphane precursor tosulforaphane. In some embodiments, the composition comprises more thanone enzyme capable of converting the sulforaphane precursor tosulforaphane. In some embodiments wherein the methods or compositionscomprise more than one enzyme, the above amounts may refer to the amountof each enzyme, or the total amount of the enzymes.

The present invention also provides for the use of a broccoli extractand/or powder, including but not limited to broccoli seed and sproutextracts and powders. The present invention provides methods ofadministration of broccoli extract and/or powder, and compositionscomprising broccoli extract and/or powder. In some embodiments, thebroccoli extract or powder is standardized to contain about 1 to about75% w/w, more preferably about 2.5% to about 50%, even more preferablyabout 5% to about 25%, and most preferably about 10% to about 20% of asulforaphane precursor, preferably glucoraphanin. Examples of broccoliextracts and powders include but are not limited to those described inU.S. Pat. Nos. 5,411,986; 5,725,895; 5,968,505; 5,968,567; 6,177,122;6,242,018; 6,521,818; 7,303,770, and 8,124,135, each of which isincorporated by reference in its entirety. Powders of broccoli may beobtained, for example, by air drying, freeze drying, drum drying, spraydrying, heat drying and/or partial vacuum drying broccoli, preferablybroccoli sprouts. In some embodiments, the compositions and methodscomprise use of about 1 μg to about 10 g, more preferably about 250 μgto about 5 g, even more preferably about 500 μg to about 1 g, preferablyabout 600 μg to about 500 mg, more preferably about 750 μg to about 400mg, and most preferably about 1 mg to about 300 mg of the broccoliextract. In some embodiments, the broccoli extract or powder is presentin a composition or administered to a subject in amounts sufficient toprovide a sulforaphane precursor or sulforaphane in the amountsdescribed above. In some embodiments, the composition may furthercomprise an enzyme potentiator, preferably ascorbic acid. In someembodiments, the method may further comprise administration of an enzymepotentiator, preferably ascorbic acid.

The sulforaphane or a derivative thereof, the sulforaphane precursor,and/or the enzyme capable of converting the sulforaphane precursor tosulforaphane may be obtained from any source, including but not limitedto one or more plants from the Brassicaceae (also known as Cruciferae)family. Examples of plants from the Brassicaceae family include, but arenot limited to, the following: broccoli, Brussels sprouts, cauliflower,cabbage, horseradish, parsnip, radish, wasabi, watercress, and whitemustard. In some preferred embodiments, sulforaphane precursor,preferably glucoraphanin, and the enzyme, preferably myrosinase, areobtained from broccoli, broccoli sprouts, or broccoli seeds. Thesulforaphane precursor and the enzyme may be obtained from the samesource or from different sources. In some embodiments, both thesulforaphane precursor and the enzyme may be obtained from an extract orpowder from these plants, preferably a broccoli seed or sprout extractor powder.

The present invention provides for the use of an enzyme potentiator.Enzyme potentiators may be used to enhance the activity of the enzymethat is capable of converting the sulforaphane precursor tosulforaphane. In some embodiments, the enzyme potentiator comprises anenzyme co-factor, preferably ascorbic acid. Ascorbic acid, also known asascorbate or vitamin C, can potentiate the activity of myrosinase. Insome embodiments, without an enzyme potentiator such as ascorbic acid,the conversion reaction to sulforaphane may be too slow to occur in thelocation needed for peak absorption. The enzyme potentiator may beobtained from a natural source, or it may be produced synthetically.

In some embodiments, the compositions may comprise about 1 mg to about500 mg, preferably about 1 mg to about 250 mg, and most preferably about1 mg to about 125 mg of the enzyme potentiator. In some preferredembodiments, compositions suitable for human use comprise about 1 mg toabout 50 mg of the enzyme potentiator.

In some embodiments, the method of the present invention comprisesadministration of an enzyme potentiator, preferably ascorbic acid, in anamount of about 1 mg to about 500 mg, preferably about 1 mg to about 250mg, and most preferably about 1 mg to about 125 mg. In some preferredembodiments wherein the subject is a human, the method comprisesadministration of about 1 mg to about 50 mg. In some embodiments, themethod of the present invention comprises administration of the enzymepotentiator, preferably ascorbic acid, in an amount of about 0.01 mg/kgto about 3 mg/kg, and most about 0.02 mg/kg to about 2 mg/kg. In somepreferred embodiments wherein the subject is a human, the methodcomprises administration of about 0.02 mg/kg to 0.7 mg/kg of the enzymepotentiator. The above amounts may refer to each dosage administrationor a total daily dosage.

In some embodiments, the method comprises administration of more thanone enzyme potentiator. In some embodiments, the composition comprisesmore than one an enzyme potentiator. In some embodiments wherein themethod or composition comprises the use of more than one enzymepotentiator, the above amounts may refer to the amount of each enzymepotentiator, or the total amount of the enzyme potentiators.

The present invention further comprises magnesium or a salt or complexthereof. The present invention provides for the use of any compoundscontaining magnesium, such as magnesium oil. Salts of magnesium include,but are not limited to: magnesium sulfate, magnesium oxide, magnesiumcitrate, magnesium glutamate, magnesium gluconate, magnesium glycinate,magnesium bromide, magnesium carbonate, magnesium chloride, magnesiumfluoride, magnesium iodide, magnesium nitrate, magnesium perchlorate,magnesium permanganate, magnesium phosphate, magnesium oratate,magnesium malate, magnesium aspartate, and dimagnesium malate. Complexesof magnesium include, but are not limited to magnesium chelates, such asmagnesium bisglycinate chelate magnesium lysinate glycinate chelate,magnesium glycinate glutamine chelate. In some preferred embodiments,the magnesium or a salt or complex thereof comprises magnesium sulfate,magnesium oxide, magnesium citrate, magnesium malate, magnesiumglycinate, dimagnesium malate, magnesium bisglycinate chelate, ordimagnesium malate. In some embodiments, the compositions thecompositions and/or methods of the present invention comprise magnesiummalate, or magnesium bisglycinate chelate, which is a form ofbioavailable magnesium which presents a low adverse effect and druginteraction profile with a high level of absorption. In someembodiments, the compositions and/or methods comprise the use of morethan one magnesium or salt or complex thereof.

In some embodiments, magnesium or a salt or complex thereof may be used.In some embodiments, the composition of the present invention comprisesabout 1 to about 1000 mg, preferably about 5 mg to about 750 mg, morepreferably about 10 mg to about 500 mg, and most preferably about 15 mgto about 350 mg. In some preferred embodiments wherein the compositionsare suitable for human use, the composition comprises about 20 mg toabout 200 mg of elemental magnesium.

In some embodiments, the method of the present invention comprisesadministration of about 1 mg to about 1000 mg, preferably about 5 mg toabout 750 mg, more preferably about 10 mg to about 500 mg, and mostpreferably about 15 mg to about 350 mg. In some preferred embodimentswherein the subject is a human, the method comprises administration ofabout 20 mg to about 200 mg of elemental magnesium. In some embodiments,the method of the present invention comprises administration of about0.1 mg/kg to about 15 mg/kg, preferably about 0.15 mg/kg to about 10mg/kg, more preferably about 0.2 mg/kg to about 7.5 mg/kg, morepreferably about 0.3 mg/kg to about 5 mg/kg, and most preferably about0.3 mg/kg to about 4 mg/kg. In some preferred embodiments wherein thesubject is a human, the method comprises administration of about 0.3mg/kg to about 3 mg/kg of elemental magnesium. The above amounts mayrefer to each dosage administration or a total daily dosage.

In some embodiments, the method comprises administration of more thanmagnesium or a salt or complex thereof. In some embodiments, thecomposition comprises more than one type of magnesium or a salt orcomplex thereof. In some embodiments wherein the method or compositioncomprises more than one type of magnesium or a salt or complex thereof,the above amounts may refer to the amount of each magnesium or a salt orcomplex thereof, or the total amount of the magnesium or a salt orcomplex thereof.

In some embodiments, a vitamin K2 may be further used. The addition ofvitamin K2, which is optional, may provide a synergistic effect. VitaminK2, which is also known as menaquinone, can be provided in the form ofmenaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6),menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9),menaquinone-10 (MK-10), menaquinone-11 (MK-11), and phylloquinone.Phylloquinone can be obtained from plant sources such as green leafyvegetables and has a short half-life in the plasma, but it can beconverted to menaquinone-4 (MK-4) by the endothelium, testes andpancreas. It can be synthesized by intestinal bacteria and is also foundin cheeses. In some preferred embodiments, vitamin K2 is providedthrough menaquinone-7 (MK-7).

In some embodiments, the composition comprises about 10 μg to about 500μg, preferably about 20 μg to about 400 μg, more preferably about 40 μgto about 300 μg, and most preferably about 50 μg to about 250 μg ofmenaquinone (MK-7). In some preferred embodiments wherein thecomposition is suitable for human use, the composition comprises about75 μg to about 250 μg of menaquinone-7 (MK-7).

In some embodiments, the method comprises administration of about 10 μgto about 500 μg, preferably about 20 μg to about 400 μg, more preferablyabout 40 μg to about 300 μg, and most preferably about 50 μg to about250 μg of menaquinone-7 (MK-7). In some preferred embodiments whereinthe subject is a human, the method comprises administration of about 75μg to about 250 μg of menaquinone-7 (MK-7) to a subject. In someembodiments, the method comprises administration of about 0.1 μg/kg toabout 8 μg/kg, preferably about 0.3 μg/kg to about 5 μg/kg, morepreferably about 0.5 μg/kg to about 4.5 μg/kg, and most preferably about0.75 μg/kg to about 3.5 μg/kg of menaquinone-7 (MK-7) to a subject. Insome preferred embodiments wherein the subject is a human, the methodcomprises administration of about 1 μg/kg to about 3 μg/kg ofmenaquinone-7 (MK-7) to a subject. The above amounts may refer to eachdosage administration or a total daily dosage.

Each of the components of the compositions and methods of the presentinvention, for example, the sulforaphane precursor, the enzyme capableof converting the sulforaphane precursor to sulforaphane, the enzymepotentiator, and/or magnesium or a salt or complex thereof may beobtained from a natural source or produced synthetically.

The methods of the present invention may further comprise administrationof one or more additional components. The compositions of the presentinvention may further comprise one or more additional components. Theadditional components may include active pharmaceutical ingredients,nutritional supplements, and nutritional extracts. Examples ofadditional components include, but are not limited, glucan, ursolicacid, quercetin or a derivative thereof, an aminosugar such asglucosamine, a glycosaminoglycan such as chondroitin, avocado/soybeanunsaponifiables, vitamins such as vitamin K2, coffee fruit, magnesium,ursolic acid, proanthocyanidins, alpha- and beta-glucans, curcumin,phytosterols, phytostanols, and S-adenosylmethionine (SAMe). Theseadditional components may be present in milk thistle (Silybum marianum)extract (silymarin), cranberry (Vaccinium macrocarpon) extract(proanthocyanidins, quercetin, and ursolic acid), turmeric (Curcumalonga), medicinal mushrooms (such as maitake shiitake, or reishimushrooms).

In some embodiments, the ratio of magnesium or a salt or complex thereofto sulforaphane or a derivative thereof is about 1:1 to about 50:1,preferably about 1.5:1 to about 20:1, more preferably about 1.75:1 toabout 15:1, more preferably about 2:1 to about 11:1, and most preferablyabout 2:1 to about 8:1. In some embodiments, the ratio of magnesium or asalt or complex thereof to sulforaphane precursor is about 1:1 to about25:1, preferably about 2:1 to about 10:1, more preferably about 3:1 toabout 8:1, more preferably about 4:1 to about 7:1, and most preferablyabout 4:1 to about 6:1.

In some embodiments, the composition comprises a unit dosage form,including but not limited to pharmaceutical dosage forms suitable fororal, rectal, intravenous, subcutaneous, intramuscular, transdermal,transmucosal, and topical. In some preferred embodiments, thecomposition comprises an orally administrable dosage form or a rectallyadministrable dosage form. Examples of orally administrable dosage formsinclude, but are not limited to a tablet, capsule, powder that can bedispersed in a beverage, a liquid such as a solution, suspension, oremulsion, a soft gel/chew capsule, a chewable bar, or other convenientdosage form known in the art. In preferred embodiments, the compositioncomprises a tablet, capsule, or soft chewable treat. The orallyadministrable dosage forms may be formulated for immediate release,extended release or delayed release.

In some embodiments, at least the sulforaphane precursor, the enzyme,and the enzyme potentiator are provided in a dosage form which allowsfor the release in an area of the gastrointestinal tract having a pH ofat least 4 and preferably at least 5, such as the small intestine,preferably the duodenum. In some embodiments, at least the sulforaphaneor derivative thereof and/or the broccoli extract or powder are providedin a dosage form which allows for the release in an area of thegastrointestinal tract having a pH of at least 4 and preferably at least5, such as the small intestine, preferably the duodenum. In someembodiments, the magnesium or a salt or complex thereof and/or anyoptional additional components are also released in an area of thegastrointestinal tract having a pH of at least 4 and preferably at least5, such as the small intestine, preferably the duodenum. The smallintestine includes the duodenum, jejunum, and ileum.

In some embodiments, each of these components (i.e, sulforaphaneprecursor, enzyme, enzyme potentiator, sulforaphane or a derivativethereof, broccoli extract or powder, magnesium or a salt or complexthereof, and/or additional components) are released simultaneously orconcomitantly (i.e., within a short period of time of each other). Thisprovides benefits over glucoraphanin-containing compositions formulatedto release the glucoraphanin in an area of the gastrointestinal tracthaving a pH below 4, such as the stomach. In low pH environments such asthis, the acidic environment may divert conversion of sulforaphaneprecursor to other, physiologically inactive end products, such assulforaphane nitrile and epithionitrile.

In some embodiments, the compositions may comprise orally administrablecompositions which comprise gastroprotective formulations, includingenteric coated dosage forms or any dosage form which is resistant todegradation in an area of the gastrointestinal tract having pH below 4,such as the stomach. For example, the orally administrable compositionmay comprise a tablet or capsule comprising an enteric coating. Theenteric coating may comprise materials including, but not limited tocellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,polyvinyl acetate phthalate, methacrylic acid copolymer, methacrylicacid:acrylic ester copolymer, hydroxypropyl methylcellulose acetatesuccinate, hydroxypropyl methylcellulose trimellitate, shellac,cellulose acetate trimellitate, carboxymethylethylcellulose, andmixtures thereof. The enteric coating may comprise any suitable entericpolymers known in the art. In some embodiments, one or more of thecomponents in the composition may be embedded in a matrix of entericpolymers. In some embodiments, the orally administrable compositionscomprise a capsule that dissolves slowly in gastric acid and travels tothe small intestine, such as DRCAPS™ acid resistant capsules, which aremarketed by CAPSUGEL® or any other acid resistant capsules.

In the most preferred form, the orally administrable composition issurrounded by a coating that does not dissolve unless the surroundingmedium is at a pH of at least 4, and more preferably at least 5.Alternatively, a coating may be employed which controls the release bytime, as opposed to pH, with the rate adjusted so that the componentsare not released until after the pH of the gastrointestinal tract hasrisen to at least 4, and more preferably at least 5. Thus, atime-release formulation may be used to prevent gastric presence of thesulforaphane precursor, the enzyme capable of converting thesulforaphane precursor to sulforaphane, and the enzyme potentiator, orof the sulforaphane. The coating layer(s) may be applied onto orallyadministrable composition using standard coating techniques. The entericcoating materials may be dissolved or dispersed in organic or aqueoussolvents. The pH at which the enteric coat will dissolve can becontrolled by a polymer, or combination of polymers, selected and/orratio of pendant groups. For example, dissolution characteristics of thepolymer film can be altered by the ratio of free carboxyl groups toester groups. Enteric coating layers also contain pharmaceuticallyacceptable plasticizers such as triethyl citrate, dibutyl phthalate,triacetin, polyethylene glycols, polysorbates or other plasticizers.Additives such as dispersants, colorants, anti-adhering and anti-foamingagents may also be included.

The compositions may contain one or more non-active pharmaceuticalingredients (also known generally as “excipients”). Non-activeingredients, for example, serve to solubilize, suspend, thicken, dilute,emulsify, stabilize, preserve, protect, color, flavor, and fashion theactive ingredients into an applicable and efficacious preparation thatis safe, convenient, and otherwise acceptable for use. The excipientsare preferably pharmaceutically acceptable excipients. Examples ofclasses of pharmaceutically acceptable excipients include lubricants,buffering agents, stabilizers, blowing agents, pigments, coloringagents, flavoring agents, fillers, bulking agents, fragrances, releasemodifiers, adjuvants, plasticizers, flow accelerators, mold releaseagents, polyols, granulating agents, diluents, binders, buffers,absorbents, glidants, adhesives, anti-adherents, acidulants, softeners,resins, demulcents, solvents, surfactants, emulsifiers, elastomers andmixtures thereof.

In some embodiments, the combination of (i) a sulforaphane precursor,preferably glucoraphanin, (ii) an enzyme capable of converting thesulforaphane precursor to sulforaphane, preferably a glucosidase enzyme,more preferably a thioglucosidase enzyme, and most preferablymyrosinase, (iii) an enzyme potentiator, preferably an enzyme co-factor,more preferably ascorbic acid, and (iv) magnesium or a salt or complexthereof demonstrates a synergistic effect. In some embodiments, thecombination of sulforaphane (or a derivative thereof) and magnesium or asalt or complex thereof demonstrates a synergistic effect. Synergyrefers to the effect wherein a combination of two or more componentsprovides a result which is greater than the sum of the effects producedby the agents when used alone. In preferred embodiments, the synergisticeffect is greater than an additive effect. In some embodiments, thecombination of a sulforaphane precursor, an enzyme capable of convertingthe sulforaphane precursor to sulforaphane, an enzyme potentiator, andmagnesium or a salt or complex thereof has a statistically significant,greater effect compared to: (i) each component alone, (ii) thecombination of sulforaphane precursor and the enzyme alone; and/or (iii)the combination of sulforaphane precursor, the enzyme, and the enzymepotentiator alone.

In preferred embodiments, the combination of the sulforaphane precursor,the enzyme, the enzyme potentiator, and magnesium or a salt or complexthereof demonstrates synergy by having a statistically significantand/or greater than additive effect compared to the sulforaphaneprecursor alone and magnesium or a salt or complex thereof alone. Insome embodiments, the combination of glucoraphanin, myrosinase, ascorbicacid, and magnesium or a salt or complex thereof has a synergisticeffect compared to the combination of glucoraphanin, myrosinase,ascorbic acid alone; and compared to magnesium.

In some embodiments, the combination of a sulforaphane (or a derivativethereof) and magnesium or a salt or complex thereof has a statisticallysignificant and/or greater than additive effect than: (i) sulforaphane(or a derivative thereof) alone, and/or (ii) magnesium or a salt orcomplex thereof alone. In some embodiments, the combination ofsulforaphane and glucan has a synergistic effect compared tosulforaphane alone, and magnesium alone.

In some embodiments, the combination of broccoli extract or powder andmagnesium or a salt or complex thereof has a statistically significantand/or greater than additive effect than: (i) broccoli extract or powderalone, and/or (ii) magnesium or a salt or complex thereof alone. In someembodiments, the combination of broccoli extract or powder and MK-7 hasa synergistic effect compared to broccoli extract or powder alone, andmagnesium alone.

The present invention provides methods of use, including methods ofadministration to a subject in need thereof. In some embodiments, themethod comprises administration of the combination of a sulforaphaneprecursor, an enzyme capable of converting the sulforaphane precursor tosulforaphane, an enzyme potentiator, and magnesium or a salt or complexthereof. In some embodiments, the method comprises administration of thecombination of a sulforaphane or a derivative thereof and magnesium or asalt or complex thereof. In some embodiments, the method comprisesadministration of the combination of a broccoli extract or powder andmagnesium or a salt or complex thereof.

In some embodiments, the methods relate to treating, preventing,reducing the occurrence of, decreasing the symptoms associated with,and/or reducing secondary recurrences of diseases or conditions such asthose associated with the endothelium and cardiovascular system in asubject. In some preferred embodiments, the diseases and conditionscomprise atherosclerosis, ischemic heart disease, acute coronarysyndrome, or arterial injury. The methods may also relate to decreasingendothelial C-Reactive Protein to reduce vascular inflammation,increasing endothelial nitric oxide to improve vasodilitation forimproved blood flow, decreasing atherosclerotic plaque calcification,reducing vascular damage or stiffness, and/or increasing bone mineraldensity.

In some embodiments, the combination may be administered for decreasinglevels or decreasing gene expression of interleukin-8 (IL-8) and/ormonocyte chemoattractant protein-1 (MCP-1) in a subject. In someembodiments, the combination may be administered for treating,preventing, reducing the occurrence of, decreasing the symptomsassociated with, and/or reducing secondary recurrences of a disease orcondition associated with elevated levels of interleukin-8 (IL-8) and/ormonocyte chemoattractant protein-1 (MCP-1) in a subject.

Examples of diseases and conditions include, but are not limited to:atherosclerosis, inflammatory bowel disease, inflammatory lung disease,chronic liver disease such as cirrhosis, inflammatory rheumatic disease,osteoarthritis, gingivitis, asthma, psoriasis, Alzheimer's disease,ischemic heart disease, acute coronary syndrome, arterial injury,arteriogenesis, depression, type II diabetes, metabolic syndrome,colorectal cancer, migraines, asthma, renal disease, osteoporosis, lymedisease, ischemic disorders, neuropathy, gastrointestinal disease, andconditions occurring specifically in animals such as laminitis (e.g., inequines) and after heart worm treatments (e.g., in dogs and cats). Thecompositions may also be administered after surgery.

In some embodiments, the methods relate to providing a beneficial effecton biomarkers, and treating, preventing, reducing the occurrence of,decreasing the symptoms associated with abnormal levels of thesebiomarkers. Examples of such biomarkers include, but are not limited toNADPH-dependent enzymes, thioredoxin (TXN), thioredoxin reductase-1(Txnrd-1), glutamate-cysteine ligase subunit (GCLC), sulfotransferase1A1 (SULT1A1), heme oxygenase-1 (HMOX1), glutathione peroxidase-3(GPx-3), glutathione S-transferse theta 2 (GSTT2), microsomalglutathione S-transferase 1 (MGST1), aldehyde oxidase (AOX1), aldo-ketoreductase 1B8 (Akr1 b8), flavin-containing monooxygenase 2 (FMO2), Fcreceptor region receptor III (Fcgr3), tryptase beta 1 (TPSB1), mast cellprotease-6 (Mcpt6), neurexin-1-alpha (NRXN-1), microphthalmia-associatedtranscription factor (MITF), type II iodothyronine deiodinase (DIO2),angiopoietin-14 (Angpt14), cluster of differentiation (CD36), and NteI.Diseases or conditions associated with elevated or abnormal levels ofthese biomarkers include, but are not limited to cancer, pulmonary andcentral nervous system tuberculosis, multiple sclerosis, Crohn'sdisease, atherosclerosis, osteoarthritis, asthma, stroke, emphysema,diabetic nephropathy, chronic histiocytic intervillositis of theplacenta, hypertension, abdominal aortic aneurysm, inflammatory boweldisease, chronic rhinosinusitis, coronary artery disease, and kidneydisease.

In some embodiments, the method comprises administering to a subject inneed thereof a combination of sulforaphane and magnesium or a salt orcomplex thereof. In some embodiments the method comprises administeringto a subject in need thereof a combination of broccoli extract or powderand magnesium or a salt or complex thereof. In some preferredembodiments, the method comprises administering to the subject acombination of glucoraphanin, myrosinase, ascorbic acid, and magnesiumor a salt or complex thereof. In preferred embodiments, the combinationsdemonstrate a synergistic effect in the methods of the presentinvention.

In preferred embodiments, one or more components of the combinations(for example, the sulforaphane precursor, the enzyme capable ofconverting the sulforaphane precursor to sulforaphane, the enzymepotentiator, the magnesium or a salt or complex thereof; or thesulforaphane or derivative thereof and the magnesium or a salt orcomplex thereof; or the broccoli extract or powder and the magnesium ora salt or complex thereof) are administered together in one compositionor dosage form, or separately, preferably within a period in which theirtherapeutic properties overlap. In some embodiments, the components ofthe combinations may be administered in two or more orally administrablecompositions or dosage forms. For example, in some embodiments, thesulforaphane precursor, the enzyme capable of converting thesulforaphane precursor to sulforaphane, and the enzyme potentiator areadministered in one orally administrable dosage form, while themagnesium or a salt or complex thereof are administered in one or moreseparate or additional orally administrable dosage form(s). In preferredembodiments, the components of the combination are administered in onedosage form.

In some embodiments, the combination may be administered at a frequencyof 1 to 10 times daily, preferably 1 to 5 times daily, more preferably 1to 3 times daily, and most preferably 1 time daily.

The dosages disclosed in this application refer generally to dosagessuitable for humans. Dosage calculations can be determined by those ofskilled in the art by evaluating body weight, surface area, metabolicrate, and species differences.

The term “subject” refers to any animal, including mammals and birds.Mammals include, but are not limited to, humans, dogs, cats, horses,cows, camels, elephants, lions, tigers, bears, seals, and rabbits. Inpreferred embodiments, the subjects comprise mammals that are notconsumed as food, such as humans, cats, and dogs.

EXAMPLES Example 1

The following is an exemplary formulation:

Glucoraphanin-containing broccoli seed extract (about 12% w/w), 50 mg to5 gramsMyrosinase-containing freeze-dried broccoli sprout powder, 25 mg to 500mgAscorbic acid, 5 mg to 500 mgMagnesium malate, providing 20 to 200 mg elemental magnesium.

Example 2

A Hydrophobic Interaction Chromatographic (HILIC) method was developed,comprising the following conditions:

Column: Waters BEH Amide, 1.7-μm particle size; 2.1 mm×100 mm

Mobile Phase: 20% 10 mM Ammonium Acetate, pH 5.0; 80% Acetonitrile;

Separation mode: isocratic

Column Temperature: 70° C.

Flow Rate: 0.7 mL/minThe above conditions allow separation of five typical Brassicaceaeglucosinolates, including the sulforaphane precursor, glucoraphanin.

Example 3 Consumption of Glucoraphanin as a Function of the AscorbicAcid Concentration

About 250 mg of broccoli seed extract containing about 12% (w/w)glucoraphanin were subjected to hydrolysis by a fixed concentration ofbroccoli sprout-derived myrosinase in the presence of variableconcentration of ascorbic acid, ranging from 0 to 600 μmoles/Liter. Thereaction mixtures were thermostated at 38° C.; aliquots were withdrawnevery 15 minutes for 60 minutes, and concentration of glucoraphanindetermined chromatographically. The rate of glucoraphanin consumptionwas interpreted as the rate its conversion to sulforaphane. Graphicalrepresentation of glucoraphanin content reduction as a function ofincreasing ascorbic acid concentration results in a series of linearplots; the slopes of the linear regression lines reflect the rate ofglucoraphanin consumption, in μmoles/minute. It is apparent that in thepresence of 600 μmoles/Liter concentration of ascorbic acid, thereaction rate increased 13-fold relative to that which proceeded in theabsence of modulatory effects of ascorbic acid.

Content of Ascorbic Acid 250 μM Time, min 0 μM 50 μM 125 μM 250 μMFiltered 400 μM 600 μM 0 93.36 93.36 93.36 93.36 93.36 93.36 93.36μmoles 15 92.24 89.20 84.52 80.95 86.31 78.32 75.02 GR 30 90.71 84.2475.92 69.06 79.44 62.78 55.66 45 89.44 80.30 68.09 57.63 71.94 47.6737.50 60 87.79 76.36 59.41 45.76 65.18 33.15 22.09 Slope −0.09293−0.28599 −0.56217 −0.79012 −0.47140 −1.00714 −1.20029 μmol/min Intercept93.496 93.271 93.123 93.053 93.386 93.270 92.734 μmol

Example 4 Equimolar Conversion of Glucoraphanin to Sulforaphane

A two-part experiment was conducted to further elucidate the role ofascorbic acid in modulating myrosinase activity. All solutions wereprepared in 20 mM Tris-buffered saline, at pH 7.5, previously identifiedas an optimal for myrosinase activity; each sample tube had 100 mg offreeze-dried broccoli powder accurately weighed in as a source ofmyrosinase. Experiment was conducted at 38° C. for 2 hours, with samplealiquots removed in 30-minute increments, and both glucoraphanin andsulforaphane content assessed by HPLC. A strongly acidic “stop” solutionwas utilized to instantaneously inhibit further myrosinase activity inthe removed aliquots. A control sample contained no ascorbic acid, andthe enzymatic conversion proceeded unassisted by a co-factor.

PART 1. In the presence of the fixed concentration of ascorbic acid, 1mmol/Liter, an increasing amount of broccoli seed extract (about 12%glucoraphanin, w/w) was added, ranging from 250 mg to 500 mg.

PART 2. While keeping the amount of broccoli seed extract fixed at 250mg, the concentration of ascorbic acid was varied from 0.4 mmol/Liter to3.8 mmol/Liter.

The table below presents glucoraphanin and sulforaphane expressed inμmoles. It is apparent that within the first 30 minutes in almost allthe reaction mixtures, conversion of glucoraphanin to sulforaphane wascomplete. However, careful examination of the enzymatic conversionoccurring in the control sample, without the stimulating effects ofascorbic acid, reveals an equimolar conversion of glucoraphanin tosulforaphane, i.e., the amount of glucoraphanin consumed results in theequivalent amount of sulforaphane produced.

Glucoraphanin, μmoles Sulforaphane, μmoles Time, min 0 30 60 90 120 0 3060 90 120 GR 250 mg AA 0.0 mM 58.02 48.57 37.52 26.58 15.67 3.42 12.0822.27 33.17 42.89 GR 250 mg AA 1.0 mM 40.07 21.51 61.95 60.20 60.0458.25 GR 300 mg AA 1.0 mM 49.31 24.18 74.40 73.04 72.19 70.56 GR 350 mgAA 1.0 mM 61.41 25.00 84.92 84.02 83.19 80.02 GR 400 mg AA 1.0 mM 71.351.56 26.71 96.60 95.38 93.39 91.16 GR 500 mg AA 1.0 mM 89.41 1.01 33.52120.16 118.45 116.45 112.34 GR 250 mg AA 0.4 mM 45.66 15.98 62.06 61.0160.88 58.90 GR 250 mg AA 1.0 mM 35.24 26.49 62.19 60.62 60.41 59.10 GR250 mg AA 2.0 mM 24.94 36.05 60.85 59.78 59.65 58.08 GR 250 mg AA 2.9 mM22.24 38.20 59.95 59.34 58.77 56.99 GR 250 mg AA 3.8 mM 21.70 37.8758.77 57.79 58.41 56.17

In the Part 2 of the experiment, the modulatory effect of the increasingconcentration of ascorbic acid on the activity of myrosinase wasassessed. An initial, apparently linear, increase in myrosinase-promotedconversion of glucoraphanin to sulforaphane is observed to about 2mmol/L of ascorbic acid concentration, followed subsequently by aconsiderable leveling off.

Finally, examination of sulforaphane yield of after 30 minutes withinthe PART 1 of the experiment, reveals that in the presence of 1mmol/Liter of ascorbic acid, the fixed amount of myrosinase contained in100 mg of freeze-dried broccoli sprout powder is capable of generatingat least 200 μmoles of sulforaphane, in a predictably linear fashion.FIGS. 1, 2, 3, and 4 demonstrate the results of this study.

Example 5 Conversion of Glucoraphanin to Sulforaphane in the Presence ofSimulated Intestinal Fluid

Simulated Intestinal Fluid (SIF) powder, a commercially suppliedconcentrate closely approximating the human intestinal content in termsof composition, pH and ionic strength, was used. The experiment utilizeda USP Dissolution Apparatus 2 (paddles), where into six dissolutionvessels 500 mL of Simulated Intestinal Fluid was dispensed, along with150 mg of freeze-dried broccoli sprout powder as a source of myrosinase.In vessels 1-4, the concentration of ascorbic acid was varied from 0.25to 1.00 mmol/Liter; in vessel 5, in addition to 1 mmol/Liter ascorbicacid, 3.125 g of pancreatin (8×USP) was suspended; in vessel 6, inaddition to 1 mmol/Liter ascorbic acid, and 3.125 g of pancreatin(8×USP), a doubled amount of freeze-dried broccoli sprout powder (300mg) was added. After vessels were brought to 38° C., 250 mg ofglucoraphanin-rich (8%, w/w) broccoli seed extract was added to each,and the resulting suspensions were stirred at 75 RPM for 2 hours.Aliquots were withdrawn every 15 minutes, and assayed for sulforaphane.FIG. 4 shows direct correlation between larger yield of sulforaphane andhigher concentrations of ascorbic acid, especially at the earlier stagesof the experiment.

Example 6

The following study was conducted to determine the effect of thecombination of sulforaphane and magnesium sulfate (MgSO₄) on expressionof monocyte chemoattractant protein (MCP-1), which is a mediator ofinflammation. MCP-1 is chemokine which attracts monocytes, macrophages,and lymphocytes to sites of inflammation. A reduction in MCP-1 isbeneficial for the health of endothelial cells which line thecardiovascular system.

In the study, RAW cells were activated with 1 μg/mL lipopolysaccharide(LPS) for 30 minutes to stimulate the induction of MCP-1 geneexpression. Following LPS treatment, the cells were treated with one ofthe following: (i) DMSO (vehicle control), (ii) 0.5 μM SFN, (iii) 2.5 mMMgSO₄, or (iv) the combination of 0.5 μM SFN and 2.5 mM MgSO₄. Aftertreatment for 24 hours, the MCP-1 levels were assessed via quantitativeRT-PCR.

The results, which are shown in FIG. 5, demonstrate that the combinationof sulforaphane and magnesium sulfate had a synergistic (greater thanadditive effect) compared to sulforaphane and magnesium sulfate alone.The data shows that the following reduction in MCP-1 levels: magnesiumsulfate alone resulted in an approximately 16% decrease, sulforaphanealone resulted in an approximately 29% decrease, and the combination ofmagnesium sulfate and sulforaphane resulted in an approximately 53%decrease. This shows that the combination had a greater than additiveeffect in reducing MCP-1 levels.

Example 7

A subject presents with atherosclerosis and is suffering from symptomsincluding damage to the arteries and chest pain. She is administered atablet containing glucoraphanin, myrosinase, ascorbic acid, andmagnesium malate. The tablet is an enteric coated formulation whichreleases the contents in the small intestine. After one month of dailyadministration of the tablet, the subject experiences modulation ofsurrogate biomarkers including interleukin-8 (IL-8) and/or monocytechemoattractant protein-1 (MCP-1) which correlate with improvement insymptoms.

1. An orally administrable composition comprising: a sulforaphaneprecursor; an enzyme capable of converting the sulforaphane precursor tosulforaphane; an enzyme potentiator; and magnesium or a salt or complexthereof.
 2. The orally administrable composition of claim 1, wherein thesulforaphane precursor comprises glucoraphanin.
 3. The orallyadministrable composition of claim 1, wherein the enzyme capable ofconverting the sulforaphane precursor to sulforaphane comprisesmyrosinase.
 4. The orally administrable composition of claim 1, whereinthe enzyme potentiator comprises ascorbic acid.
 5. The orallyadministrable composition of claim 1, wherein the composition comprisesan enteric-coated dosage form.
 6. The orally administrable compositionof claim 1, wherein the composition further comprises one or moreadditional components is selected from the group consisting of: vitaminK2, quercetin, an aminosugar, a glycosaminoglycan, avocado/soybeanunsaponifiable, a vitamin, coffee fruit, silymarin, proanthocyanidins,ursolic acid, curcumin, phytosterols, and phytostanols.
 7. (canceled) 8.The orally administrable composition of claim 1, comprisingglucoraphanin, myrosinase, ascorbic acid, and a magnesium salt.
 9. Theorally administrable composition of claim 1, wherein the compositioncomprises broccoli extract or powder.
 10. A method of treating,preventing, reducing the occurrence of, decreasing the symptomsassociated with, and reducing secondary recurrences of a cardiovascularcondition or disorder, comprising administering to a subject in needthereof a sulforaphane precursor; an enzyme capable of converting thesulforaphane precursor to sulforaphane; an enzyme potentiator; andmagnesium or a salt or complex thereof.
 11. The method of claim 10,wherein the sulforaphane precursor comprises glucoraphanin.
 12. Themethod of claim 10, wherein the enzyme capable of converting thesulforaphane precursor to sulforaphane comprises myrosinase.
 13. Themethod of claim 10, wherein the enzyme potentiator comprises ascorbicacid.
 14. The method of claim 10, comprising administration ofglucoraphanin, myrosinase, ascorbic acid, and a magnesium salt.
 15. Themethod of claim 10, comprising administering an enteric-coated dosageform.